The document compares the biocompatibility of aluminosilicate samples containing iron and either dysprosium or yttrium. Scanning electron microscopy images show morphological changes on the sample surfaces after incubation in a collagen solution. Attenuated total reflectance Fourier-transform infrared spectroscopy analysis and deconvolution of the amide I region indicate qualitative and quantitative differences in the secondary structure of adsorbed collagen compared to native collagen. Specifically, collagen adsorbed to the yttrium aluminosilicate sample showed a more pronounced modification of secondary structure, indicating lower biocompatibility. Cyclic voltammetry further supported the quantitative investigations by showing enhanced current intensity and decreased oxidation potential of collagen adsorbed to the electrode surface

1. JOURNAL OF OPTOELECTRONICS AND ADVANCED MATERIALS - SYMPOSIA, Vol. 2, No. 1, 2010, p. 140 - 144
Comparison between nanostructured aluminosilicate
systems with yttrium/dysprosium and iron: structural
investigation and biocompatibility evaluation
S. CAVALU*
, F. BANICA, V. SIMONa
University of Oradea, Faculty of Medicine and Pharmacy, Oradea 410087 Romania
a
Babes-Bolyai University, Faculty of Physics & Institute for Interdisciplinary Experimental Research, Cluj-Napoca
400084, Romania
The biocompatibility evaluation of aluminosilicate samples containing iron and dysprosium or yttrium was made with respect
to collagen (type I from calf skin) adsorption. The SEM analysis indicates morphological changes on samples surface after
incubation in collagen solution. At the same time, the features of ATR-FTIR spectra and the data obtained by deconvolution
of the amide I region of adsorbed collagen show qualitative and quantitative diferences compared to the native protein. The
secondary structure of collagen is more pronounced modified upon adsorption to yttrium aluminosilicate indicating a lower
biocompatibility compared to dysprosium containing sample. Cyclic voltammetry also supports the quantitative
investigations by collagen adsorption at the Ag/AgCl electrode surface. The current intensity enhancement and the
decrease of the oxidation potential of collagen indicate that collagen adsorption is an irreversible process.
(Received April 21, 2009; accepted October 1, 2009)
Keywords: Aluminosilicates, SEM, ATR-FTIR, Cyclic voltammetry
1. Introduction
Aluminosilicate glasses with iron and
yttrium/dysprosium incorporated investigated in this study
are of great interest in the treatment of degenerative
diseases by hyperthermia and radiotherapy, because they
could be used in internal therapy of cancer, both by
hyperthermia and local irradiation of the malignant
tumours with high energy and short range beta radiation
[1, 2]. The ferromagnetic nanoparticles developed in the
vitroceramic biomaterial cause heating through hysteresis
losses or magnetic relaxation phenomena and can induce
the necrosis of the tumours. On the other hand, the yttrium
and dysprosium stable isotopes can be activated by
neutron irradiation to radioactive isotopes which have
convenient properties for cancer radiotherapy [3, 4].
Beside the melt undercooling method used to obtain
aluminosilicate systems, the sol-gel synthesis was also
tacken into account [5].
The primer condition imposed to materials considered
for biomedical applications is biocompatibility dictated by
the manner in which their surface interact with blood
constituents (erythrocytes, platelets) as well as the proteins
[6, 7]. The type and amounts of adsorbed proteins mediate
subsequent adhesion, proliferation and differentiation of
cells as well as depositing of mineral phases. The
behaviour of a protein at an interface is likely to differ
considerably from its behaviour in the bulk. Because of the
different local environment at the interface, the protein
may have the opportunity of adopting a more disordered
state exposing its hydrophobic core to the aqueous phase,
often called surface denaturation. Denaturation is a process
by which hydrogen bonds, hydrophobic interactions and
salt linkages are broken and the protein is unfolded. The
denaturation of secondary structure involves also changes
in ratio among the three common structures: α helix, β
sheets or turns and unordered [8, 10]. FTIR spectroscopy
can be used to study protein secondary structure in any
state, i.e. aqueous, frozen, dried or even as an insoluble
aggregate, and for this reason it is one of the most used
techniques for studying stress induced alterations in
protein conformation and for quantifying protein
secondary structure. ATR-FTIR can provide important
information leading to the development of novel
biomaterials as replacements for damaged or diseased
natural tissue. The spectral region of amide I (1660 cm-1
),
amide II (1550 cm-1
) and amide III (1300cm-1
) are very
sensitive to the conformational changes in the secondary
structure of proteins. Computational techniques based on
the second derivative spectra and deconvolution procedure
is used for percentage evaluation of each secondary
structure and also the perturbations upon the adsorption to
different surfaces [9-12]. Collagen type I is the most
abundant protein of the extracellular matrix, a fibrillar
triple helical structure that forms gel networks in irregular
connective tissue. Collagen is also proline-rich and self
assembles into fibrils [13,14].
In the present study, the biocompatibility of
aluminosilicate samples incorporating iron and
yttrium/dysprosium was evaluated with respect to collagen
adsorption. The adsorbed collagen layer on the samples
surfaces was investigated by SEM, ATR-FTIR and Cyclic
Voltammetry.
2. Materials and methods
Reagent grade silicic acid SiOx(OH)4-2x, and nitrates
Al(NO3)3·9H2O, Fe(NO3)3, Y(NO3)3 Dy(NO3)3 were used
as starting materials to prepare by sol-gel method [5]
10Dy2O3·10Fe2O3·60SiO2·20Al2O3 (DFSA) and
10Y2O3·10Fe2O3·60SiO2·20Al2O3 (YFSA) samples. The
compositions are indicated in mol%. The 110o
C dryed sol
gels were heat treated at 500°C and 1200°C. Collagen type
I from calf skin (lyophilized) was purchased from Sigma
Chemicals. All samples were separatelly incubated for 24
hours at 37 °C in 2 mg/mL collagen phosphate buffered
solution and, after filtration and drying process, the sample
surfaces were analyzed by SEM and ATR FTIR.

2. Comparison between nanostructured aluminosilicate systems with yttrium/dysprosium and iron: structural investigation ... 141
Scanning Electron Microscopy (SEM) was performed
with a JEOL JSM5510 microscope in order to study the
morphology of the surfaces, before and after incubation.
The FT-IR spectra of the samples before and after
incubation were recorded in the region 4000-600 cm-1
by a
Bruker EQUINOX 55 spectrometer OPUS software, using
an Attenuated Total Reflectance accessory with a scanning
speed of 32 cm-1
min-1
and the spectral width 2.0 cm-1
. The
internal reflection element was a ZnSe ATR plate (50 x 20
x 2 mm) with an aperture angle of 45°. A total of 128
scans were accumulated for each spectrum. Spectra were
recorded at a nominal resolution of 2 cm-1
. The spectra
were smoothed with a 9-point Savitsky–Golay smooth
function to remove the white noise. The second derivative
spectral analysis of amide I band was applied to locate
positions and assign them to different functional groups.
Before starting the fitting procedure, the obtained depths
of the minima in the second derivative spectrum and,
subsequently, the calculated maximum intensities were
corrected for the interference of all neighbouring peaks.
All second-derivative spectra, calculated with the
derivative function of Opus software, were baseline-
corrected, based on the method of Dong and Caughey
[10], and area-normalized under the second derivative
amide I region, 1700–1600 cm-1
[15]. Curve fitting was
performed by setting the number of component bands
found by second-derivative analysis with fixed bandwidth
(12 cm-1
) and Gaussian profile. The area under each peak
was used to calculate the percentage of each component
and, finally, to analyze the percentage of secondary
structure component [10,15].
Cyclic voltammetric (CV) studies were carried out
with a TraceLab 150 system, equipped with a Trace
Master interface board, in residual protein solutions. A
conventional three-electrode cell was employed
incorporating a carbon-paste working electrode (with or
without zeolite), a saturated Ag/AgCl reference electrode,
and a Pt-wire counter electrode [16]. The supporting
electrolyte solutions were 0.05 M phosphate buffer (pH 6-
8) and acetate (pH ≤5). Voltammetric experiments were
carried out in deoxygenated solutions by pure nitrogen.
Stock solutions 0.1 M were prepared by dissolving in
water the appropriate amount of each compound, usually
their potassium salts. Working solutions were prepared by
successive dilution of the stock solutions.
3. Results and discussion
The as prepared samples and those obtained by 500o
C
heat treatment are in non-crystalline state, while by the
heat treatment applied at 1200o
C nanocrystalline structures
are achieved.
In order to study the morphological details of the
samples surfaces, SEM analysis were performed before
and after immersion in collagen solution. Fig. 1 clearly
illustrates the changes occurred on YFSA and DFSA
sample surfaces after the incubation in the solution
containing collagen protein. According to the literature
[15], once the protein has covered the surface of implants,
host cells are no longer able to contact the underlying
foreign-body material but only the protein–coated surface.
The adsorbed protein layer-rather then the foreign material
itself may stimulate or inhibit further biochemical
processes.
a b c
d e
Fig. 1. The morphology of YFSA and DFSA sample surfaces before (a, b) and after incubation (c, d) along with the SEM image
of native collagen fibre (e).
ATR-FTIR spectra of both 500°C and 1200°C heat
treated samples, before and after incubation in collagen
solution, are presented in Fig. 2. The dominant bands
around 1087 cm-1
are assigned to the stretching vibration
of Si-O-Si and Al-O-Al bonds, while the Al-O stretching
vibrations of tetrahedral AlO4 groups are related with the
bands at around 789 cm-1
. Other weak absorption bands at
around 912 cm-1
are present in the spectra of the samples

3. 142 S. Cavalu, F. Banica, V. Simon
is
focu
hydrogen bonds associated with the carbonyls [19].
reported studies, along with the
q
Fig. gen
type I, used to prepare the protein solution.
Table 1. Assignment and relati dsorbed to 500o
C heat treated
uminosilicate sample ng iron and yttrium/dysprosium.
helix helix helix turns
treated at 1200°C, also attributed to the silica lattice [17].
The intensity of these bands is significantly reduced upon
incubation. One can observe that collagen is preferentially
adsorbed to the samples treated at 500°C, emphasized by
the characteristic amide I at 1624/1635 cm-1
and imide II
at 1429/1418 cm-1
. As a reference, the FTIR spectrum of
native collagen is shown in Fig. 3, pointing out the
features characteristic of amide I, II and III which are the
most intense vibrational modes. The present study
sed on the amide I behavior, which is due primarily to
the stretching vibrations of the peptide carbonyl group.
As shown in Fig. 2 (b, d), the amide bands of
adsorbed collagen are shifted towards lower wavenumber
upon adsorption (compared with the amide bands of the
native protein). According to the literature, the intensity of
amide I band of collagen decreases markedly upon
denaturation, and after deconvolution, four prominent
components are present both in the native or denaturated
protein spectrum [13,18]. That the relative intensities of
these four peaks vary with the extent of collagen-fold or
triple helix content speaks to the point that they are clearly
conformationally dependent. Specific components within
the fine structure of amide I adsorbed collagen is
correlated with different states of hydrogen bonding
associated with the local conformations of the alpha chain
peptide backbones. This heterogeneity can arise either
from intrinsic basicity differences in the strengths of the
Deconvolution of amide I band of native collagen and
adsorbed to our aluminosilicate samples with iron and
yttrium/dysprosium is shown in Fig. 4 a,b,c and the
assignment of the components in Table 1 was made on the
basis of the previous
ua e analysis.ntitativ
1800 1600 1400 1200 1000 800 600
0.000
0.025
0.050
0.075
0.100
0.125
0.150
0.175
0.200
0.225
0.250
1228
AmideIII
W avenumber cm
-1
Absorbance(a.u.)
1640
AmideI
1546
AmideII
3. ATR FTIR spectrum of the lyophilized colla
ve area of amide I components of native collagen, or respectively a
al s containi
α α αCollagen
amide I ν ν ν ν(cm-1
) A (%) (cm-1
) A (%) (cm-1
) A (%) (cm-1
) A (%)
native
collagen
1640 44.6 1653 23.5 1666 23.1 1710 8.8
ad o 1635 34.0 1640 44.0 1663 12.0 1673 10sorbed t
YFSA
ad o
DFSA
1624 40.2 1641 25.5 1657 23.5 1670 10.8sorbed t
1580 1600 1620 1640 1660 1680 1700 1720 1740
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
Wavenumber cm
-1
(a)
Absorbance(a.u.)
1600 1610 1620 1630 1640 1650 1660 1670 1680 1690
000005
000000
000005
000010
000015
000020 (b)
Wavenumber cm
-1
Absorbance(a.u.)
1610 1620 1630 1640 1650 1660 1670 1680
00002
00000
00002
00004
00006
00008
00010
00012
00014
00016
00018
(c)
Wavenumber cm
-1
Absorbance(a.u.)
Fig. 4. Deconvoluted amide I absorption band of native collagen (a) and adsorbed collagen to YFSA (b) and DFSA (c) samples.

4. Comparison between nanostructured aluminosilicate systems with yttrium/dysprosium and iron: structural investigation ... 143
mod ed upon adsorption. As a general behaviour, one can
sent a
beter behavior with respect to collagen adsorption.
Curve fits to the amide I native collagen reveals four
Gaussian components at 1640, 1653, 1666 and 1710 cm-1
representing helix-related hydrogen-bounded set of
carbonyls. According to the literature, the highest
frequency carbonyl absorption peak represents the weakest
H-bonded system [18]. Beside the characteristic
frequencies of α helix conformation, the peak located in
the higher region, at 1710 cm-1
, represent the formation of
an antiparallel β-sheet structure (or turns). Both the
intensity and the location of the characteristic peaks are
observe a shift toward lower frequencies, a decrease in α
helix content and concomitant increase of turn percentage
upon adsorption, as a consequence of denaturation.
Comparing the quantitative results in table 1, we can
remark that the sample ASY10Fe10 appear to be more
susceptible to conformational changes due to the
adsorption process, since spectral alteration reflected on
the components percentage is more obvious as compared
with the native protein. In terms of biocompatibility, we
suggest that dysprosium/iron aluminosilicate pre
ifi
500 1000 1500 2000 2500 3000
-0.08
-0.06
-0.04
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
YFSA
afterincubation
1200
o
C 789
500
o
C
Wavenumbercm
-1
Absorbance(a.u.)
1428
1624
1087
789
912
a b
500 1000 1500 2000 2500 3000
-0.10
-0.08
-0.06
-0.04
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0 1
Wavenumber cm
-1
Absorbance(a.u.)
1200°C
500°C
DSFA
after incubation1087
1418
1635
790798
c d
SA and DFSA heat treated at 500Fig. 5. ATR FTIR spectra of the samples YF °C and 1200°C, recorded before and after
incubation in collagen solution.
.25
V vs. Ag/AgCl electrode whose intensity varies directly
proportional to the collagen concentration of solution.
Cyclic voltammetry measurements were also carried
out in residual collagen solutions using a carbon paste
electrode modified with zeolite after an original method
[20]. The goal was to study the effect of zeolite/carbon
paste electrode concentration on the accumulation of
collagen. Cyclic volatmograms at different collagen
concentrations were registered with modified carbon paste
electrode (Fig. 5) exhibiting a strong anodic peak at +0
500 1000 000 2500 30001500 2
-0.04
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
1087
500
o
C
1200
o
C
YFSA
beforeincubation
Absorbance(a.u.)
798789
912
Wavenumbercm
-1
500 1000 000 2500 3000
-0.06
1500 2
-0.04
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
500
o
C
1200
o
C
DFSA
beforeincubation
Wavenum
Absorbance(a.u.)
784
855
797
1087
ber cm
-1

5. 144 S. Cavalu, F. Banica, V. Simon
0.0 0.2 0.4 0.6 0.8 1.0
DFSA/1200
YFSA/1200
DFSA/500
YFSA/500
2.5x10
-5
I(A)
E (V)
Fig. 6. The cyclic voltammograms for different residual
collagen solutions using modified carbon paste
electrodes at pH 7 and scan rate 100 mV/s.
We can observe that the current related to both heat
treated samples at 1200°C presents a higher intensity
compared to those treated at 500°C, suggesting the
preferential collagen adsorption to the last one. The
current enhancement was remarkable, and additionally, a
significant decrease in the oxidation potential of collagen
can be distinguished (more than 100 mV) when the
electrode is modified with zeolite. This behavior, which
was observed at different concentrations of collagen and at
several scan rates potential, clearly demonstrates that the
zeolite mediate the electrocatalytically properties of
collagen [20,21]. No cathodic peak was observed in the
reverse scan, indicating that the adsorption of collagen at
zeolite modified electrode is an irreversible process.
4. Conclusions
Iron and yttriun/disprosium aluminosilicate systems
prepared by sol-gel route and heat treated at 500°C and
1200°C were characterized using SEM, ATR FTIR
spectroscopy and cyclic voltammetry. The
biocompatibility of the samples was evaluated with respect
to collagen adsorption. Qualitative and quantitative
analysis of amide I features by deconvolution and curve
fitting reveals that the samples containing with iron and
disprosium present a beter behavior with respect to
collagen adsorption. SEM images reveal different degree
of collagen adsorption toward the dysprosium/yttrium
samples. Cyclic voltammetry carried out in residual
collagen solutions indicates preferential collagen
adsorption onto the samples heat treated at 500°C as an
irreversible process, that is in agreement with the ATR-
FTIR results.
Acknowledgements
The study was supported by the scientific research
project CEEX 100/2006-MATNANTECH of the
Romanian Excellence Research Program.
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*
Corresponding author: scavalu@rdslink.ro